Replacement of Tyrosine-197 and the Corresponding Tyrosine-195 to Isoleucine in Cephalosporium acremonium and Streptomyces clavuligerus Isopenicillin N Synthase

2001 ◽  
Vol 56 (9-10) ◽  
pp. 806-809
Author(s):  
Paxton Loke ◽  
Tiow-Suan Sim
Keyword(s):  
Penicillium Chrysogenum ◽  
Biosynthetic Pathway ◽  
Specific Activity ◽  
High Specific Activity ◽  
Streptomyces Clavuligerus ◽  
Amino Acid Difference ◽  
Tyrosine Residue ◽  
Cephalosporium Acremonium ◽  
Isoleucine Residue ◽  
Isopenicillin N

AbstractIsopenicillin N synthase (IPNS) is one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyses the conversion of δ-(ʟ-α-aminoadipyl)-ʟ-cysteinyl-ᴅ-valine to isopenicillin N. The IPNS from Penicillium chrysogenum 23X-80-269-37-2, a high penicillin V-producer, was found to possess an isoleucine residue instead of tyrosine at position 195. An attempt to increase the specific activity of IPNS from Cephalosporium acremonium and Streptomyces clavuligerus was undertaken by altering the corresponding tyrosine residue to an isoleucine at the corresponding location. Unfortunately, no apparent increase in specific activity was encountered when the purified mutant enzymes were analysed and thus, this amino acid difference is likely not responsible for high specific activity in IPNS.

Zur Biosynthese der Penicilline: Bildung von 5-(2-Aminoadipyl)-cysteinyl-valin in Extrakten von Penicillium chrysogenum / Formation of 5-(2-Aminoadipyl)-cysteinyl-valin by Extracts of Penicillium chrysogenum

1970 ◽  
Vol 25 (10) ◽  
pp. 1125-1129 ◽  
Author(s):  
Karl Bauer
Keyword(s):  
Penicillium Chrysogenum ◽  
Specific Activity ◽  
Solid Phase ◽  
High Specific Activity ◽  
Phase Method ◽  
Solid Phase Method

Mycel-extracts of P. chrysogenum catalyse the formation of 5- (2-aminoadipoyl) -cysteinyl-valin out of its aminoacid components.For the synthetical preparation of this compound the solid-phase method is particularly suited. The synthesis can be carried out even with very small quantities of substance and thereby allows the preparation of radioactive labelled L-5- (2-aminoadipoyl) -L-cysteinyl-L-valin of relatively high specific activity.

Ammonium repression of cephalosporin production by Streptomyces clavuligerus

1985 ◽  
Vol 31 (8) ◽  
pp. 736-743 ◽  
Author(s):  
Alfredo F. Braña ◽  
Saul Wolfe ◽  
Arnold L. Demain
Keyword(s):  
Specific Activity ◽  
Relative Proportion ◽  
Streptomyces Clavuligerus ◽  
Defined Medium ◽  
Inhibitory Effects ◽  
Rate Limiting Step ◽  
Deacetoxycephalosporin C Synthetase ◽  
Isopenicillin N Synthetase ◽  
Rate Limiting ◽  
Isopenicillin N

Production of β-lactam antibiotics took place during growth of Streptomyces clavuligerus in chemically defined medium. The specific activities of isopenicillin N synthetase ("cyclase"), isopenicillin N epimerase, and deacetoxycephalosporin C synthetase ("expandase") increased during the exponential phase of growth. Specific cephalosporin productivity during fermentation followed a similar pattern, reaching a maximum near the end of the growth phase and decaying rapidly in the stationary phase. Ammonium chloride depressed cephalosporin production, presumably as a result of repression of cyclase and expandase formation, but not of epimerase. No inhibitory effects on enzyme activity by ammonium were found. Addition of tribasic magnesium phosphate [Mg3(PO4)2∙8H2O] prevented the repression of cyclase and markedly stimulated cephalosporin production. Cephamycin C and, in smaller amounts, O-carbamoyldeacetylcephalosporin C were the only cephalosporins detected. Growth with ammonium resulted in lower titers of both compounds, and did not change the relative proportion of each. The correlation found between cephalosporin productivity and cyclase specific activity in different media suggests that formation of this enzyme may be the rate-limiting step in the pathway.

Purification of isopenicillin N synthetase from Streptomyces clavuligerus

1986 ◽  
Vol 32 (12) ◽  
pp. 953-958 ◽  
Author(s):  
Susan E. Jensen ◽  
Brenda K. Leskiw ◽  
Leo. C. Vining ◽  
Yair Aharonowitz ◽  
Donald W. S. Westlake ◽  
...  
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
High Performance Liquid Chromatographic ◽  
Inhibitory Effect ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

Isopenicillin N synthetase was purified from Streptomyces clavuligerus by sequential salt precipitation, ion-exchange and gel-filtration chromatography using both conventional open column and high-performance liquid chromatographic techniques. Material from the final purification step had a specific activity of 204.1 × 10−3 units/mg of protein which represented a 130-fold purification over the cell-free extract. The purified isopenicillin N synthetase was determined to have a molecular weight of 33 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and to have a Km of 0.32 mM with respect to its substrate δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The enzyme showed a sensitivity to thiol-specific inhibitors with N-ethylmaleimide giving the strongest inhibitory effect.

Folate synthesis in higher-plant mitochondria: coupling between the dihydropterin pyrophosphokinase and the dihydropteroate synthase activities

2002 ◽  
Vol 363 (2) ◽  
pp. 313-319 ◽  
Author(s):  
Jean-Marie MOUILLON ◽  
Stéphane RAVANEL ◽  
Roland DOUCE ◽  
Fabrice RÉBEILLÉ
Keyword(s):  
Kinetic Data ◽  
Biosynthetic Pathway ◽  
Specific Activity ◽  
Plant Mitochondria ◽  
High Specific Activity ◽  
Limiting Factor ◽  
Maximal Velocity ◽  
Higher Plant ◽  
Bifunctional Protein ◽  
Dihydropteroate Synthase

The plant enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (HPPK/DHPS) is a mitochondrial bifunctional protein involved in tetrahydrofolate synthesis. The first domain (HPPK) catalyses the pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (dihydropterin) by ATP, leading to 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (dihydropterinPPi) and AMP. The second domain (DHPS) catalyses the next step, i.e. the condensation of p-aminobenzoic acid (p-ABA) with dihydropterinPPi to give 7,8-dihydropteroate (dihydropteroate) and PPi. In the present article we studied the coupling between these two reactions. Kinetic data obtained for the HPPK domain are consistent with an ordered Bi Bi mechanism where ATP binds first and dihydropterinPPi is released last, as proposed previously for the monofunctional Escherichia coli enzyme. In the absence of p-ABA, AMP and dihydropterinPPi accumulate and negatively regulate the reaction. In the presence of p-ABA, the rates of AMP and dihydropteroate synthesis are similar, indicating a good coupling between the two reactions. DihydropterinPPi, an intermediate of the two reactions, never accumulates in this situation. The high specific activity of DHPS relative to HPPK, rather than a preferential channelling of dihydropterinPPi between the two catalytic sites, could explain these kinetic data. The maximal velocity of the DHPS domain is limited by the availability of dihydropterinPPi. It is strongly feedback-inhibited by dihydropteroate and also dihydrofolate and tetrahydrofolate monoglutamate, two intermediates synthesized downstream in the folate biosynthetic pathway. Thus the HPPK domain of this bifunctional protein is the limiting factor of the overall reaction, but the DHPS domain is a potential key regulatory point of the whole folate biosynthetic pathway.

scholarly journals Purification of isopenicillin N synthetase

1984 ◽  
Vol 222 (3) ◽  
pp. 789-795 ◽  
Author(s):  
C P Pang ◽  
B Chakravarti ◽  
R M Adlington ◽  
H H Ting ◽  
R L White ◽  
...  
Keyword(s):  
Streptomyces Clavuligerus ◽  
Protein Band ◽  
Maximum Activity ◽  
Polyacrylamide Gel ◽  
Major Protein ◽  
Synthetase Activity ◽  
Cephalosporium Acremonium ◽  
Major Protein Band ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

Isopenicillin N synthetase was extracted from Cephalosporium acremonium and purified about 200-fold. The product showed one major protein band, coinciding with synthetase activity, when subjected to electrophoresis in polyacrylamide gel. An isopenicillin N synthetase from Penicillium chrysogenum was purified about 70-fold by similar procedures. The two enzymes resemble each other closely in their Mr, in their mobility on electrophoresis in polyacrylamide gel and in their requirement for Fe2+ and ascorbate for maximum activity. Preliminary experiments have shown that a similar isopenicillin N synthetase can be extracted from Streptomyces clavuligerus.

Site-Directed Mutagenesis of Proline-285 to Leucine in Cephalosporium acremonium Isopenicillin N - Synthase Affects Catalysis and Increases Soluble Expression at Higher Temperatures

2001 ◽  
Vol 56 (5-6) ◽  
pp. 413-415 ◽  
Author(s):  
Paxton Loke ◽  
Tiow-Suan Sim
Keyword(s):  
Biosynthetic Pathway ◽  
Soluble Expression ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Cephalosporium Acremonium ◽  
E Coli ◽  
Measurable Activity ◽  
Important Enzyme ◽  
Isopenicillin N

The conversion of δ-(ʟ-α-aminoadipyl)-ʟ-cysteinyl-ᴅ-valine (ACV) to isopenicillin N is dependant on the catalytic action of isopenicillin N - synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. One of the amino acid residues suggested by the Aspergillus nidulans IPNS crystal structure for interaction with the valine isopropyl group of ACV is proline-283. Site-directed mutagenesis of the corresponding proline- 285 to leucine in Cephalosporium acremonium IPNS resulted in non-measurable activity but an increased soluble expression at higher temperatures in a heterologous E. coli host.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

Autoprothrombin C Activity from Prothrombin with Snake Venom or Trypsin

1962 ◽  
Vol 08 (03) ◽  
pp. 425-433 ◽  
Author(s):  
Ewa Marciniak ◽  
Edmond R Cole ◽  
Walter H Seegers
Keyword(s):  
Snake Venom ◽  
Thrombolytic Agent ◽  
Sodium Citrate ◽  
Specific Activity ◽  
High Specific Activity ◽  
Citrate Solution ◽  
Clotting Factors ◽  
Activation Products ◽  
Russell’S Viper ◽  
Autoprothrombin C

SummarySuitable conditions were found for the generation of autoprothrombin C from purified prothrombin with the use of Russell’s viper venom or trypsin. DEAE chromatographed prothrombin is structurally altered and has never been found to yield autoprothrombin C and also did not yield it when Russell’s viper venom or trypsin were used. Autoprothrombin C is derived from prothrombin with tissue extract thromboplastin, but not in large amounts with the intrinsic clotting factors. With the latter thrombin and autoprothrombin III are the chief activation products. Autoprothrombin III concentrates were prepared from serum and upon activation with 25% sodium citrate solution or with Russell’s viper venom large amounts of autoprothrombin C were obtained, and this was of high specific activity. Theoretically trypsin is not a thrombolytic agent, but on the contrary should lead to intravascular clotting.

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